Protein purification

2.3.3.1 Disruption of cells

E. coli cells and large amounts of eukaryotic cells were thawed in a water bath at room temperature and lysed using a cell disruptor (Constant Systems) at 2.0 kbar and 4°C. Cell debris was removed by centrifugation for 15 min at 10.000 g and 4°C.

Small amounts of eukaryotic cells used for testexpressions or transient expression were centrifuged for 10 min at 4.500 g and 4°C and resuspended in 50 mM Tris pH 7.5, 150 mM NaCl, 1 % Triton X-100, 10 % glycerol and complete protease inhibitor. Cell lysis was performed for 15 min in a sonicator water bath at room temperature. Cell debris was removed by centrifugation for 2 min at 10.000 g and 4°C. Supernatant was immediately used for further

2.3.3.2 Preparation of HEK293S GnTI¯ endoplasmatic reticulum membranes

Cells were harvest by centrifugation for 10 min at 4.500 g and 4°C and resuspended in 3 ml of ice cold 40 mM HEPES pH 7.2, 5 mM MgCl, 5 mM EGTA, complete protease inhibitor and DNAse. Cells were passaged 35 times through a 27-gauge needle in 1 ml batches on ice. Cell debris was sedimented at 1.000 g, 4°C for 10 min. Cell pellets were resuspended in buffer without DNAse and centrifuged again. The cell pellet was then resuspended in buffer containing 2.2 M sucrose and immediately applied on a sucrose density gradient.

2.3.3.3 Preparation of E.coli and HEK293S GnTI¯ membranes

The separation of membrane and soluble proteins was performed using an ultracentrifuge.

Membranes were sedimented at 150.000 g, 4°C for 60 min, resuspended and homogenized with a dounce homogenizer in 10 - 50 ml buffer (same buffers as used for cell disruption without protease inhibitor) afterwards. The supernatant was used for the purification of soluble proteins or discarded.

2.3.3.4 Solubilisation of membrane proteins

The total protein concentration was adjusted to 10 mg/ml for E.coli membranes and 2.5 mg/ml for HEK293S GnTI¯ membranes by addition of the corresponding buffer. Membrane solution was stirred while detergent was slowly added to a final detergent concentration of 1 %. The mixture was stirred for 60 min before unsolubilised material was removed by centrifugation at 150.000 g, 4°C for 60 min. The supernatant contained solubilised membrane proteins and was used for further purification.

2.3.3.5 Immobilized nickel-nitrilotriacetic acid (Ni-NTA) chromatography

For purification of His-tagged proteins by gravity-flow chromatography preloaded Ni-NTA from Quiagen (Hilden) was used. The column was equilibrated with 5 column volumes of buffer before protein solution was added to the material. To remove unspecifically bound proteins the material was first washed with 10 column volumes of buffer, followed by buffer with increasing imidazole concentration (5 mM, 10 mM, 25 mM, 50 mM) and finally 5 column volumes of buffer with 150 mM imidazole to elute the specific bound proteins. All fractions were collected for further analysis by SDS-PAGE and Western blot.

2.3.3.6 Immobilized strep-tactin affinity chromatography

Proteins expressed with a StrepII-tag were purified using the strep-tactin sepharose from IBA (Göttingen). The column material was washed with 5 column volumes of ddH2O before it was equilibrated with the same volume of buffer. The protein sample was loaded on the column and unspecifically bound proteins were washed away with 10 column volumes of buffer and another 10 column volumes of buffer containing 500 mM NaCl. Bound proteins were eluted with buffer supplemented with 5 mM desthiobiotin.

2.3.3.7 Immobilized amylose affinity chromatography

Proteins fused to a maltose binding protein (MBP) via a TEV cleavage site were purified using an amylose resin (NEB, Frankfurt am Main). The resin was first washed with 3 column volumes of ddH2O before it was equilibrated with buffer. The protein solution was loaded on the column using gravity flow and unbound proteins were washed away using 12 column volumes of buffer.

The target protein was either eluted together with the fusion protein using buffer containing 10 mM maltose or eluted alone by the cut-off of MBP using 10 - 50 µg TEV-protease.

2.3.3.8 Immobilized lentil lectin chromatography

Proteins expressed in HEK293S GnTI¯ cells containing glycosylated extracellular domains were purified using Lentil Lectin Sepharose 4B (GE Healthcare, Freiburg). The column material was equilibrated with buffer before protein solution was added and incubated with the material over night at 4°C. The day after the column was washed with 5 column volumes of buffer containing 500 mM NaCl followed by buffer with increasing α-D-methylglucoside concentration (5 mM, 10 mM, 25 mM, and 50 mM). Glycosylated proteins were eluted with buffer containing 150 mM α-D-methylglucoside. All fractions containing the target protein were combined and used for further purification.

2.3.3.9 Anion exchange (AEX) chromatography

Before loading on an AEX chromatography column, the protein sample was dialyzed against buffer without salt and concentrated to a final volume of 5 ml if necessary. For anion exchange chromatography a Mono Q 5/50 GL column (GE Healthcare, Freiburg) was used on an Ettan system. The column was equilibrated with 50 mM HEPES pH 7.5 and the sample was loaded using a flow rate of 0.25 ml/min. The protein was eluted over 10 column volumes with a

2.3.3.10 Size exclusion chromatography (SEC)

For size exclusion chromatography an Äkta explorer, Äkta purifier or an Ettan system (GE Healthcare, Freiburg) was used. Depending on the size and amount of the construct either a Superose 6 10/300, Superose 6 PC 3.2/30, Superose 6 3.2/300, Superdex 200 10/300, Superdex 200 5/150 or Superdex 75 10/300 was used. All buffers used in a SEC were filtered and degassed before they were connected to the system. Columns were washed with ddH2O before they were equilibrated with buffer. Protein samples were concentrated to 50 - 100 µl for the Ettan system or to 500 µl for the Äkta systems and commonly centrifuged at 5.000 g for 60 s through a Spin-X filter with 0.22 µm membrane pore size (Sigma-Aldrich, Taufkirchen). The protein sample was loaded with a flow rate of either 0.03 ml/min or 0.4 ml/min depending on the column and system used for the SEC.

2.3.3.11 Sucrose density gradient centrifugation

Sucrose density gradients were used for further protein purification or for the purification of endoplasmatic reticulum (ER) membranes. Protein solution was added on top of a continuous sucrose gradient (1.0 M - 0.7 M sucrose in the corresponding buffer) and centrifuged in a swing-out rotor (SW 40 Ti or SW 60 Ti) at 200.000 g for 17 h. The gradient was separated in 25 fractions of equal size and analysed by SDS-PAGE.

A continuous sucrose gradient (1.5 M - 0.7 M sucrose in 40 mM HEPES pH 7.2, 5 mM MgCl, 5 mM EGTA and complete protease inhibitor) was added on top of disrupted cells resuspended in buffer containing 2.2 M sucrose. Membranes were separated by centrifugation for 20 h at 40.000 g at 4°C.

2.3.3.12 Concentration of protein samples

Protein samples were concentrated using Vivaspin 4 or Vivaspin 500 (Sartorius, Göttingen) centrifugal filter units with a molecular weight cut-off of 10 - 100 kDa. The samples were concentrated according to manufacture’s instruction. The centrifugation speed was set to 3.500 g.

2.3.3.13 Exchange of detergent to amphipol A8-35

Amphipol A8-35 was added to a membrane protein solution at a 3-fold excess (w/w) and the mixture was incubated at 4°C for 4 h under rocking or rotation. 15 mg of activated biobeads were added per 1 ml protein-detergent-amphipol mixture and incubated over night at 4°C.

Biobeads were removed over a plastic column. To separate single proteins in amphipol A8-35